Team:SCU-China/Linkage

From 2014.igem.org

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</td>
</td>
</tr>
</tr>
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</table><p>For &#8220;Traditional&#8221; Assembly, we set up the following system (20 &#956;l):</p><table><tr><td><p>Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)</p>
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</table><table class="table table-striped"> <caption>For &#8220;Traditional&#8221; Assembly, we set up the following system (20 &#956;l):</caption><tr><td><p>Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)</p>
</td><td><p>About 100 ng</p>
</td><td><p>About 100 ng</p>
</td>
</td>
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</table>
</table>
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<p>For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows:</p><table class="table table-striped"> <caption>PCR system (For 50&#956;L)</caption> <thead><tr><th><p>Materials</p>
 
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</th><th><p>Volume</p>
 
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</th>
 
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</tr></thead> <tbody><tr><td><p>Taq DNA polymerase</p>
 
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</td><td><p>0.5&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>Template</p>
 
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</td><td><p>0.5&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>Forward Primer</p>
 
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</td><td><p>1&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>Reverse Primer</p>
 
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</td><td><p>1&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>dNTPs (2.5mM)</p>
 
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</td><td><p>4&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>10x Buffer (Mg2+ free)</p>
 
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</td><td><p>5&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>MgCl2 (25mM)</p>
 
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</td><td><p>3&#956;L</p>
 
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</td>
 
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</tr><tr><td><p>Sterilized diluted water</p>
 
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</td><td><p>35&#956;L</p>
 
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</td>
 
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</tr></tbody>
 
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</table><p>Note: The content of template should be less than 500ng for the 50&#956;L system. For all of our experiments, the content of template of 0.5&#956;L DNA lower than 500ng.</p>
 
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<table class="table table-striped"> <caption>PCR protocol</caption><thead><tr><th><p>Temperature</p>
 
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</th><th><p>Times</p>
 
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</th>
 
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</tr></thead> <tbody><tr><td><p>95&#8451;</p>
 
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</td><td><p>5min.</p>
 
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</td><th rowspan="3">30-35 Times</th>
 
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</tr><tr><td><p>95&#8451;</p>
 
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</td><td><p>30sec.</p>
 
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</td>
 
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</tr><tr><td><p>55-65&#8451;</p>
 
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</td><td><p>30sec.</p>
 
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</td>
 
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</tr><tr><td><p>72&#8451;</p>
 
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</td><td><p>1-2min.</p>
 
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</td>
 
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</tr><tr><td><p>72&#8451;</p>
 
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</td><td><p>10min.</p>
 
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</td>
 
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</tr></tbody>
 
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</table><p>Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute.</p>
 
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</div>
 
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</div></div>
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Revision as of 22:18, 16 October 2014

Linkage Protocol

According to the advice of IGEM Competition, there are two linkage methods that we use in experiment. The first one is “3A” Assembly and the second is “Traditional” Assembly.

For “3A” Assembly, we set up the following system (20 μl):

Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)

About 100 ng

Insert DNA 1

The molar ratio of Insert DNA to Backbone is 3:1 to 5:1

Insert DNA 2

Same to Insert DNA 1

10 x T4 DNA Ligase Buffer

2 μl

T4 DNA Ligase

1 μl

H2O

Up to 20 μl
For “Traditional” Assembly, we set up the following system (20 μl):

Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)

About 100 ng

Insert DNA

The molar ratio of Insert DNA to Backbone is 3:1 to 5:1

10 x T4 DNA Ligase Buffer

2 μl

T4 DNA Ligase

1 μl

H2O

Up to 20 μl

Sichuan university

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